ROLES OF DIFFERENT FORMS OF CYTOCHROME-P450 IN THE ACTIVATION OF THE PROMUTAGEN 6-AMINOCHRYSENE TO GENOTOXIC METABOLITES IN HUMAN LIVER-MICROSOMES

We reported previously that the potent mutagen 6-aminochrysene is catalyzed principally by rat liver microsomal P4501A and P4502B enzymes to reactive metabolites that induce umu gene expression in 0-acetyltransferase-over-expressing strain Salmonella typhimurium NM2009; the proposal was made that there are different mechanisms in the formation of reactive N-hydroxylated and diolepoxide metabolites by P450 enzymes (Yamazaki,H. and Shimada,T., Biochem. Pharmacol., 44, 913 - 920, 1992). Here we further examined the roles of human liver P450 enzymes and the mechanism of activation of 6-aminochrysene by rat and human P450 enzymes in the Salmonella tester strains. Liver microsomes from 18 different human samples catalyzed activation of 6-aminochrysene more efficiently in S.typhimurium NM2009 than in the original strain of S.typhimurium TA1535/pSK1002. The rates of 6-aminochrysene activation in 18 human liver samples showed good correlation to the contents of P4502B6 as well as contents of P4503A4 and the respective mono-oxygenase activities catalyzed by P4503A4. Among purified P450 enzymes examined, P4501A2 as well as P4503A4 were highly active in transforming 6-aminochrysene to reactive metabolites, suggesting the involvement of different human P450 enzymes in the reaction. Four human samples that contained relatively high levels of particular P450 enzymes in their microsomes were selected and used for further characterization. Liver microsomes from human samples HL-13 and HL-4 that contained the highest levels of P4502B6 and P4503A4 respectively, were sensitive to the respective antibodies raised against monkey P4502B and human P4503A4; the activity in sample HL-16 having the highest level of P4501A2 was inhibited by anti-P4501A2 IgG. Alpha-Naphthoflavone enhanced the activation of 6-aminochrysene very significantly in human liver microsomes enriched in P4503A4 and P4502B6 enzymes. Pentachlorophenol, an inhibitor of acetyltransferase activity, suppressed the activation of 6-aminochrysene in liver microsomes from phenobarbital-treated rats and from human samples HL-4, HL-13 and HL-18 but not HL-16. In contrast, 1,1,1-trichloropropane-2,3-oxide, an inhibitor of epoxide hydrolase activity, enhanced the activation of 6-aminochrysene catalyzed by liver microsomes from beta-naphthoflavone-treated rats and from human samples HL-16 but not HL-4, HL-13 and HL-18. Inclusion of purified rat epoxide hydrolase to the reconstituted system containing rat and human P4501A enzymes caused a decrease in the rates of 6-aminochrysene activation. These results suggested that 6-aminochrysene can be metabolized to reactive metabolites by several human P450 enzymes including P4501A2, P4502B6 and P4503A4 and that there are different mechanisms of 6-aminochrysene activation by several P450 enzymes. We propose that the P450 enzymes belonging to the 1A subfamily catalyze activation of 6-aminochrysene through the formation of epoxide intermediates, while P4502B and possibly P4503A enzymes activate through an N-hydroxylation pathway.